Prolactin (PRL) secretion by the anterior pituitary (AP) is responsive to changes in physiological conditions and many external factors that also affect brain neurosteroid levels. This study tested the hypothesis that neurosteroids can affect PRL secretion in sheep under basal, stressful and advanced pregnancy conditions. In Experiment 1, luteal-phase sheep were subjected to a three-day series of intracerebroventricular (icv.) control (n = 12) or allopregnanolone (AL, 4 × 15 μg/60 μL/30 min at 30-min intervals, n = 12) infusions. Acute stressful stimuli, isolation and partial movement restriction were applied on the third day of infusion to half of the animals in each group. In Experiment 2, pregnant sheep were subjected to a three-day series of icv. control (n = 6) or finasteride (4 × 25 μg/60 μL/30 min at 30-min intervals, n = 6) infusions during the 16th week of pregnancy. As a result, the relative abundance of PRL transcript increased in the AP of luteal-phase sheep treated with stress, AL and AL in combination with stress (P < 0.05 - P < 0.01) compared to controls. The level of PRL mRNA in stressed-AL-treated sheep was higher (P < 0.01) than in sheep only subjected to stress. The PRL protein content in the AP decreased in stressed-only sheep compared to controls (P < 0.05) and increased in stressed-AL-treated sheep compared to controls and other groups (P < 0.05 - P < 0.01). Plasma PRL concentration increased (P < 0.05 - P < 0.01) in stressed-only sheep compared to controls; AL infusion counteracted the stress-induced increase in PRL levels (P < 0.05 - P < 0.01) and had no effect in non-stressed animals. Inhibition of neurosteroid synthesis in the brain of pregnant sheep by finasteride caused transient increases (P < 0.05 - P < 0.001) in plasma PRL concentration compared to controls. In conclusion, the presented results indicate a bimodal effect of AL on PRL secretion in sheep: first at the molecular level – stimulation of PRL mRNA expression; second – inhibition of hormone release from pituitary lactotrophs. Both AL activities may involve various mechanisms regulating PRL secretion. In general, cerebral neurosteroids can affect the supply of pituitary PRL in the body under certain conditions, such as stress and pregnancy.